The USRDS lists diabetic glomerulosclerosis (DG) as the leading cause of ESRD in the United States. The observation that 35-40% of the estimated 20 million Americans with diabetes (DM) develop DG, is indicative that this complication develops in a subset of genetically at risk individuals. The wild type (WT) p66ShcA gene has emerged as a genetic determinant of longevity, that controls mitochondrial metabolism, and cellular responses to oxidative stress, aging and apoptosis. The major objective of this proposal will be to determine if deletion of WTp66ShcA from the genome of mutant Akita mice, confers an oxidant resistant phenotype that protects resident glomerular cells from reactive oxygen species (ROS) dependent signals that initiate and promote progression of DG and target genomic DNA, activating genetic programs for apoptosis and cell senescence. We have proposed a model in which silencing WTp66ShcA or deleting WTp66ShcA from the Akita genome by homologous recombination with p66ShcA-/- mouse, results in the translocation of the potent stress response regulator FOXO3a to the nucleus, where it orchestrates the stress response program. The focus of Specific Aim 1 will be the identity of the molecular components of this novel stress response program. The experimental approach will employ in vitro cell culture system of conditionally immortalized human podocytes to test if silencing WTp66ShcA can rescue this terminally differentiated, highly specialized cell population from hyperglycemia-induced ROS death signal. Mutations at the bradykinin 1 and 2 receptor loci (B1/B2-/-) have been reported to increase the risk and severity of DG and aging phenotypes in diabetic Akita mice. Under Specific Aims 2 &3, WTp66ShcA will be deleted from the genome of Akita mice, to test if this loss of function mutation induces sustained renoprotection in DM, by inhibiting the generation and transmission of ROS danger signals that inflict irreversible injury to the GFB, trigger glomerular remodeling and target genomic DNA, inducing cell entry to apoptosis and senescence programs. The proposed studies are fundamental to the development of gene based strategies with the goal of to arresting or preventing DG. PUBLIC HEALTH RELEVANCE Multiple lines of evidence indicate genetic susceptibility and oxidative stress (ROS) are critical factors in development of diabetic nephropathy. The p66ShcA protein plays a pivotal role in the generation of hyperglycemic ROS signals that drive the pathobiology of diabetic nephropathy and inflict irreversible cell injury. The central hypothesis of this application is homozygous mutation at the p66ShcA locus, in mice genetically at risk for diabetic nephropathy, will attenuate or prevent biomarkers of incipient diabetic nephropathy and ROS phenotypes of apoptosis and cell senescence.